Project Summary. Cytochrome P450 (P450 or CYP) enzymes constitute a superfamily of heme proteins that metabolize a wide variety of both endogenous and exogenous substrates and play key roles in many biological processes. Only recently has it become clear that normal and pathological visual functions are among these. A growing body of evidence suggests the importance of cholesterol in the retina for normal vision. However, little is currently known about cholesterol turnover in the retina and the significance of cholesterol-hydroxylating P450s in retinal function. In extraocular tissues, P450 enzymes initiate all quantitatively important pathways of cholesterol degradation and are crucial for maintenance of overall cholesterol homeostasis. Two cholesterol-metabolizing P450s (27A1 and 46A1) were recently found to be highly expressed in the retina. CYP27A1 is a polyfunctional ubiquitous sterol 27-hydroxylase extensively characterized by us and others. CYP46A1 is a recently cloned and therefore much less studied enzyme. We began to work with CYP46A1 only four years ago. It is known that CYP46A1 controls cholesterol elimination from the brain, and its deficiency may contribute to Alzheimer's disease. We have already established that CYP46A1 is an unusual cholesterol-metabolizing P450: it seems to have very broad substrate specificities that overlap in part with those of CYP27A1 and a dual subcellular localization. Expression in specific cell types suggests that CYPs 46A1 and 27A1 play unique roles in the retina. To begin to delineate these roles as well as the metabolic fate of cholesterol in the retina, we need to know more about CYP46A1 and perform kinetic characterization of the two enzymes against overlapping substrates. In this new grant application we propose to: 1) characterize substrate specificities of CYP46A1 and compare them with those of CYP27A1;2) define the CYP46A1 active site;and 3) ascertain the significance of CYP46A1 subcellular location in retinal function. The results will allow prediction of the in vivo retinal substrates for CYP46A1 and help to understand whether CYPs 46A1 and 27A1 play the same roles in the retina as they do in extraretinal tissues. Characterization of CYP46A1 will also provide invaluable information for our on-going comparative analysis of cholesterol-metabolizing P450s and will lead to a better understanding of the mechanisms that control substrate specificity and catalytic efficiency in this set of P450s. Studies with cholesterol analogs, kinetic experiments using purified enzymes, computer modeling, site-directed mutagenesis, crystallographic, and biophysical'methods will be used to achieve the goals of the project. Relevance (for lay audience). This research will be critical for understanding the development of certain eye diseases that reduce vision.